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Ignicoccus hospitalis forms many cell surface appendages, the Iho670 fibers (width, 14 nm; length, up to 20 [micro]m), which constitute up to 5% of cellular protein. They are composed mainly of protein Iho670, possessing no homology to archaeal flagellins or fimbrins. Their existence as structures different from archaeal flagella or fimbriae have gone unnoticed up to now because they are very brittle. doi: 10.1128/JB.00858-09

Pyrococcus furiosus ('rushing fireball') was named for the ability of this archaeal coccus to rapidly swim at its optimal growth temperature, around 100[degrees]C. Early electron microscopic studies identified up to 50 cell surface appendages originating from one pole of the coccus, which have been called flagella. We have analyzed these putative motility organelles and found them to be composed primarily (>95%) of a glycoprotein that is homologous to flagellins from other archaea. Using various electron microscopic techniques, we found that these flagella can aggregate into cable-like structures, forming cell-cell connections between ca. 5% of all cells during stationary growth phase. P. furiosus cells could adhere via their flagella to carbon-coated gold grids used for electron microscopic analyses, to sand grains collected from the original habitat (Porto di Levante, Vulcano, Italy), and to various other surfaces. P. furiosus grew on surfaces in biofilm-like structures, forming microcolonies with cells interconnected by flagella and adhering to the solid supports. Therefore, we concluded that P. furiosus probably uses flagella for swimming but that the cell surface appendages also enable this archaeon to form cable-like cell-cell connections and to adhere to solid surfaces.

The novel nylon-flocked swab and a corresponding protocol to recover Bacillus spore concentrations on stainless steel and other surfaces are evaluated. The results have contributed to the cleanliness control of future life detection missions and have provided improvement in the detection of Bacillus anthracis contamination for law enforcement and security efforts.

The cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein are analyzed during assembly operations. The multiassay cultivation approach is used for the detection of several bacteria that is not cultivated from these spacial environments before and has also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus.

Different methods for storage of macroorganismic material on board ships or at remote locations were tested and how each performed with respect to survival and recoverable diversity of bacteria were evaluated. It is concluded that SD and SGT methods for storage of macroorganismic material result in equal or higher survival of total bacteria and allow recovery of a higher diversity of bacteria than the other three methods tested.

Newly erupted black smokers (hydrothermal vent chimneys) are sterile during their formation, but house hyperthermophilic microorganisms in substantial amounts in later stages. No direct experimental data exist by which mechanisms hyperthermophiles colonize newly erupted black smokers, but a scenario was proposed recently how this might happen. Here we combine high temperature light microscopy with electron microscopy to show that two hyperthermophilic Archaea, namely Pyrococcus furiosus and Methanocaldococcus villosus are able to adhere onto authentic black smoker material (BSM). We especially are able to directly observe the adhesion process via video recordings taken at high temperatures. These data validate the hypothesis that hyperthermophiles are transferred by serendipitous water currents to the outside of newly formed black smokers and react within seconds to the there prevailing high temperatures by very fast movements. They scan the surface of the hydrothermal chimneys via a much slower zigzag seek-movement and adhere via their flagella at a suitable place, building up biofilms. [ABSTRACT FROM AUTHOR]

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea. [ABSTRACT FROM AUTHOR]

The bacterial flagellar apparatus, which involves ~40 different proteins, has been a model system for understanding motility and chemotaxis. The bacterial flagellar filament, largely composed of a single protein, flagellin, has been a model for understanding protein assembly. This system has no homology to the eukaryotic flagellum, in which the filament alone, composed of a microtubule- based axoneme, contains more than 400 different proteins. The archaeal flagellar system is simpler still, in some cases having ~13 different proteins with a single flagellar filament protein. The archaeal flagellar system has no homology to the bacterial one and must have arisen by convergent evolution. However, it has been understood that the N-terminal domain of the archaeal flagellin is a homolog of the N-terminal domain of bacterial type IV pilin, showing once again how proteins can be repurposed in evolution for different functions. Using cryo-EM, we have been able to generate a nearly complete atomic model for a flagellar-like filament of the archaeon Ignicoccus hospitalis from a reconstruction at ~4-Å resolution. We can now show that the archaeal flagellar filament contains a β-sandwich, previously seen in the FlaF protein that forms the anchor for the archaeal flagellar filament. In contrast to the bacterial flagellar filament, where the outer globular domains make no contact with each other and are not necessary for either assembly or motility, the archaeal flagellin outer domains make extensive contacts with each other that largely determine the interesting mechanical properties of these filaments, allowing these filaments to flex. [ABSTRACT FROM AUTHOR]

Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492) and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized. In this work, we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI) named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA) and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mitomycin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least seven protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we identified a total of 47 asntDNA- associated GIs that were classified into 12 groups of homology differing in the encoded functionalities but sharing with GIE492 a conserved recombination module and potentially its mobility features. Most of these GIs encoded factors with proven or potential role in pathogenesis, constituting a major reservoir of virulence factors in this species. [ABSTRACT FROM AUTHOR]

The swimming behavior of Bacteria has been studied extensively, at least for some species like Escherichia coli. In contrast, almost no data have been published for Archaea on this topic. In a systematic study we asked how the archaeal model organisms Halobacterium salinarum, Methanococcus voltae, Methanococcus maripaludis, Methanocaldococcus jannaschii, Methanocaldococcus villosus, Pyrococcus furiosus, and Sulfolobus acidocaldarius swim and which swimming behavior they exhibit. The two Euryarchaeota M. jannaschii and M. villosus were found to be, by far, the fastest organisms reported up to now, if speed is measured in bodies per second (bps). Their swimming speeds, at close to 400 and 500 bps, are much higher than the speed of the bacterium E. coli or of a very fast animal, like the cheetah, each with a speed of ca. 20 bps. In addition, we observed that two different swimming modes are used by some Archaea. They either swim very rapidly, in a more or less straight line, or they exhibit a slower kind of zigzag swimming behavior if cells are in close proximity to the surface of the glass capillary used for observation. We argue that such a "relocate-and-seek" behavior enables the organisms to stay in their natural habitat [ABSTRACT FROM AUTHOR]

The model organism Pyrococcus furiosus has recently been reported to interact with Methanopyrus kandleri in coculture, suggesting a H2 symbiosis. In the current study we further investigated this hypothesis by growing P. furiosus with four other hyperthermophilic methanogens providing evidence that the organisms did not only exert positive effects (P. furiosus/Methanocaldococcus villosus and P. furiosus/Methanocaldococcus infernus) on each other, but also neutral (P. furiosus/Methanocaldococcus jannaschii) and even inhibitory interactions (P. furiosus/Methanotorris igneus) were detected suggesting interspecies relationships not only based on H2 symbiosis. Using various microscopic techniques we further analyzed the coculture with the highest positive interactions (P. furiosus/ M. villosus) concerning its growth behavior on various surfaces, which turned out to be in stark contrast to the previous reported coculture of P. furiosus/M. kandleri. This communication provides new insights into possible interactions of extremophilic Archaea in cocultures and again raises the question if and how hyperthermophilic Archaea communicate besides metabolic intermediates like H2. [ABSTRACT FROM AUTHOR]

The surfaces of 8 bacterial and 23 archaeal species, including many hyperthermophilic Archaea, could be stained using succinimidyl esters of fluorescent dyes. This allowed us for the first time to analyze the mode of cell wall growth inArchaea by subculturing stained cells. The data obtained show that incorporation of new cell wall material in Archaca follows the pattern observed for Bacteria: in the coccoid species Pyrococcus furiosus incorporation was in the region of septum formation while for the rod-shaped species Methanopyrus kandleri and Methanothermus sociabilis, a diffuse incorporation of cell wall material over the cell length was observed. Cell surface appendages like fimbriae/pili, fibers, or flagella were detectable by fluorescence staining only in a very few cases although their presence was proven by electron microscopy. Our data in addition prove that Alexa Fluor dyes can be used for in situ analyses at temperatures up to 100°C. [ABSTRACT FROM AUTHOR]

In the course of this biodiversity study, the cultivable microbial community of European spacecraft-associated clean rooms and the Herschel Space Observatory located therein were analyzed during routine assembly operations. Here, we focused on microorganisms capable of growing without oxygen. Anaerobes play a significant role in planetary protection considerations since extraterrestrial environments like Mars probably do not provide enough oxygen for fully aerobic microbial growth. A broad assortment of anaerobic media was used in our cultivation strategies, which focused on microorganisms with special metabolic skills. The majority of the isolated strains grew on anaerobic, complex, nutrient-rich media. Autotrophic microorganisms or microbes capable of fixing nitrogen were also cultivated. A broad range of facultatively anaerobic bacteria was detected during this study and also, for the first time, some strictly anaerobic bacteria (Clostridium and Propionibacterium) were isolated from spacecraft-associated clean rooms. The multiassay cultivation approach was the basis for the detection of several bacteria that had not been cultivated from these special environments before and also led to the discovery of two novel microbial species of Pseudomonas and Paenibacillus. [ABSTRACT FROM AUTHOR]

Presents information on the transfer of genetic elements by enterococcus faecalis. Information on the genetic traits that are passed; Details on an experiment conducted.

The sex pheromone system of Enterococcus faecalis was discovered by observing a clumping reaction of E. faecalis strains during conjugative transfer of plasmids. It was found that only a special type of E. faecalis plasmids, the so-called sex pheromone plasmids, are transferred via this mechanism. Various experiments, especially by the group of D. B. Clewell, led to the formulation of a model describing how the sex pheromone system works. Small linear peptides, the so-called sex pheromones, are excreted by strains not processing the corresponding sex pheromone; they react to the presence of the peptide by production of a plasmid-encoded adhesin, the so-called aggregation substance. This adhesin allows contact between the non-motile mating partners; after conjugative transfer of the plasmid, the former recipient possesses and replicates the new plasmid. Thereby the population of E. faecalis strains is shifted to a high percentage of donor strains. This is especially true because a donor strain will still excrete sex pheromones corresponding to plasmids it does not harbor; therefore, such a strain can also function as recipient for other sex pheromone plasmids it does not posses. Various aspects of this unique plasmid collection mechanism have been studied during the last few years. The data indicate that, with the exception of pAM373, all sex pheromone plasmids posses one DNA region which is highly similar to and codes for the adhesin. It is also becoming more and more clear that regulatory functions/proteins are not conserved between different sex pheromone plasmids. Induction of adhesin synthesis needs the action of a regulatory cascade composed of unique features; at the moment we are just beginning to understand this cascade. By sequencing might act also as an adhesin for eucaryotic cells, probably by interaction with integrins. At least in the case of the in vitro cultured pig kidney tubulus cell line LLC-PK1 this idea could be verified. An interesting aspect of the sex pheromone system op E. faecalis is its evolution. I will discuss the idea that two different components, both of which well might contribute to virulence of the opportunistic pathogenic bacterium, were combined in the species of E. faecalis to result in this unique plasmid collection system. [ABSTRACT FROM AUTHOR]

The expression of the structural genes for the protein synthesis initiation factor 3 (IF-3), threonyl-tRNA synthetase and phenylalanyl-tRNA synthetase carried by the transducing phage λp2 was studied in a DNA-dependent transcription-translation system in vitro and the results were compared to the regulatory pattern in vivo. In vitro, the DNA of the phage λp2 gives rise to the formation of the two forms of IF-3 (IF-31 and IF-38) which are known to be present in vivo. The kinetics of synthesis indicate an interconversion of IF-31 into IF-38. Addition of excess purified IF-31 does not significantly repress IF-3 synthesis but does stimulate the rate of conversion of IF-31 into IF-38. This apparent lack of autoregulation in vitro is in accordance with gene-dosage-dependent synthesis in vivo. The fact that strains with more than one copy of the IF-3 structural gene contain a higher relative amount of IF-38, than do haploid ones suggests that the proteolytic conversion of IF-31 into IF-38 may occur predominantly in the free (non-ribosome-bound) state. In vivo, the amount of IF-3 varies with the growth rate muck like elongation factor Tu or aminoacyl-tRNA synthetases. As with the aminoacyl-tRNA synthetases, IF-3 synthesis is not significantly subject to a stringent control system. This coordinated regulatory response in vivo, however, is not paralleled by the susceptibility of synthesis in vitro to guanosine 3'-diphosphate (ppGpp), since IF-3 formation is inhibited by ppGpp whereas that of theonyl-tRNA synthetase and phenylalanyl-tRNA synthetase is stimulated. [ABSTRACT FROM AUTHOR]

Identifies the factors modulating the transcription and translation of ribosomal protein S20 in vitro and isoleucyl-transfer RNA synthetase from Escherichia coli. Stimulation of S20 synthesis; Dihydrodipicolinic acid reductase; Carbamoyl-phosphate synthetase; Formation of amino acid activating enzymes; Rates of ribosomal assembly.

Sex-pheromone-plasmid-bearing strains of Enterococcus faecalis react with sex pheromone to induce the expression of an adhesin, the so-called aggregation substance, on their cell surface. Here we show that, by complementation studies, for sex-pheromone plasmid pAD1, expression of the structural gene asa1, coding for an aggregation substance, is mediated by a diffusible factor encoded on pAD1. We were able to demonstrate that a small open reading frame, traE1,is sufficient for transcription of the operon containing asa1. A model for expression of asa1 under the influence of the positive regulator is presented, which is supported by our observation that regulation involves an all-or-nothing induction phenomenon, leading to cells either fully expressing asa1 or not at all. [ABSTRACT FROM AUTHOR]

The sex-pheromone system of Enterococcus faecalis can be viewed as a unique and highly efficient plasmid-collection mechanism. The contact needed for transfer of the conjugative sex-pheromone plasmids is mediated by an adhesin, called aggregation substance, which is encoded by these plasmids. We show here that for 17 of the 18 sex-pheromone plasmids (pAM373 being the exception) described to date, their adhesins are immunologically related to each other. In each case, we observed the presence of an N-terminal fragment of about 78 kDa in addition to the 137-kDa form of mature aggregation substance. The cross-reactions were different for the various plasmids. In the case of pPD1 the 78-kDa fragment reacted only weakly. The aggregation substance encoded by sex-pheromone plasmid pAD1 (Asa1) was characterized in detail. The conditions used for SDS/PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asa1 might be a glycoprotein. Antibodies were isolated which are directed against the N- and C-terminal parts of aggregation substance. They showed about the same reactivity on Western blots; however, only antibodies directed against the N-terminal part of the aggregation substance could inhibit the bacterial cell/cell contact. The reactions of the two antibody preparations with induced cells of E. faecalis was analyzed by transmission electron microscopy. The results indicated that especially the N-terminal par1 of aggregation substance is exposed on the cell surface of E. faecalis; the C-terminal part seems to be much less exposed. [ABSTRACT FROM AUTHOR]

The Iho670 fibers of the hyperthermophilic crenarchaeon of Ignicoccus hospitalis were shown to contain several features that indicate them as type IV pilus-like structures. The application of different visualization methods, including electron tomography and the reconstruction of a three-dimensional model, enabled a detailed description of a hitherto undescribed anchoring structure of the cell appendages. It could be identified as a spherical structure beneath the inner membrane. Furthermore, pools of the fiber protein Iho670 could be localized in the inner as well as the outer cellular membrane of I. hospitalis cells and in the tubes/vesicles in the intermembrane compartment by immunological methods. [ABSTRACT FROM AUTHOR]

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference with life detection experiments. Therefore, space agencies measure the cultivable spore load ("bioburden") of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus spores from Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl- Eichinger, AppI. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency), was then compared to the current European Space Agency (ESA) standard wipe assay in sampling four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis). Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial motility is driven by the rotation of flagellar filaments that supercoil. The supercoiling involves the switching of coiled-coil protofilaments between two different states. In archaea, the flagellar filaments responsible for motility are formed by proteins with distinct homology in their N-terminal portion to bacterial Type IV pilins. The bacterial pilins have a single N-terminal hydrophobic α-helix, not the coiled coil found in flagellin. We have used electron cryo-microscopy to study the adhesion filaments from the archaeon Ignicoccus hospitalis. While I. hospitalis is non-motile, these filaments make transitions between rigid stretches and curved regions and appear morphologically similar to true archaeal flagellar filaments. A resolution of ~7.5Å allows us to unambiguously build a model for the packing of these N-terminal α-helices, and this packing is different from several bacterial Type IV pili whose structure has been analyzed by electron microscopy and modeling. Our results show that the mechanism responsible for the supercoiling of bacterial flagellar filaments cannot apply to archaeal filaments. [Copyright &y& Elsevier]

We analyze here for the first time the swimming behavior of a thermophilic, strictly anaerobic Spirochete, namely Spirochaeta thermophila using high temperature light microscopy. Our data show that S. thermophila very rapidly can change its morphology during swimming, resulting in cells appearing nearly linear, in cells possessing three different spiral forms, and in cells being linear at one end and spiral at the other end. In addition cells can rapidly bend by up to 180°, with their ends coming into close contact. We combine electron with light microscopy to explain these various cell morphologies. Swimming speeds for cells with the various morphologies did not differ significantly: the average speed was 33 (± 8) μm/s, with minimal and maximal speeds of 19 and 59 μm/s, respectively. Addition of gelling agents like polyvinylpyrrolidone or methyl cellulose to the growth medium resulted in lower and not higher swimming speeds, arguing against the idea that the highly unusual cell body plan of S. thermophila enables cells to swim more efficiently in gel-like habitats.